MicroRNAs (miRNAs) participate in the reproductive process by regulating the expression of target genes, however, there are few studies on the regulation of mRNA target by miRNAs in medaka (Oryzias latipes). Our previous data showed that miR-202-5p has a sex-biased expression and miR-26 is abundantly expressed in both ovary and testis of medaka. In this study, DEAD-box helicase 3, X-linked (ddx3x) was proposed to be a putative target of miR-26 and miR-202-5p through bioinformatic analysis. Here, medaka ddx3x (Olddx3x) was cloned and sequence alignment analysis indicated that Olddx3x is highly conserved and has more than 90% identity with some other teleosts. Reverse transcription polymerase chain reaction (RT-PCR) showed that Olddx3x was highly expressed in testis and ovary. Meanwhile, chromogenic in situ hybridization manifested that Olddx3x was abundantly expressed in the early oocytes in the ovary and in various stages of spermatogenesis, especially the obvious signal in the testis of spermatocytes and sperm. Dual-luciferase reporter assay and the injection of miR-26 agomir and antagomir demonstrated that the expression of Olddx3x was regulated by miR-26 but not miR-202-5p. The overall results revealed that miR-26 may perform a vital role in medaka reproduction development by regulating the expression of ddx3x.

中文翻译：

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MiR-26调节medaka（Oryzias latipes）性腺中ddx3x的表达。

微小RNA（miRNA）通过调节靶基因的表达参与生殖过程，但是，关于在medaka（Oryzias latipes）中miRNA调控mRNA靶的研究很少。我们以前的数据表明，miR-202-5p具有性别偏向性表达，而miR-26在medaka的卵巢和睾丸中均大量表达。在这项研究中，通过生物信息学分析，X连锁的DEAD-box解旋酶3（ddx3x）被提议作为miR-26和miR-202-5p的假定靶标。在这里，克隆了medaka ddx3x（Olddx3x），序列比对分析表明，Olddx3x是高度保守的，与其他硬骨鱼有90％以上的同一性。逆转录聚合酶链反应（RT-PCR）显示Olddx3x在睾丸和卵巢中高表达。与此同时，显色原位杂交表明，Olddx3x在卵巢的早期卵母细胞和精子发生的各个阶段大量表达，尤其是在精子细胞和精子的睾丸中明显的信号。双荧光素酶报告基因测定法和miR-26 agomir和antagomir的注射证明Olddx3x的表达受miR-26调控，但不受miR-202-5p调控。总体结果显示，miR-26可能通过调节ddx3x的表达在medaka繁殖发育中发挥重要作用。双荧光素酶报告基因测定法和miR-26 agomir和antagomir的注射证明Olddx3x的表达受miR-26调控，但不受miR-202-5p调控。总体结果显示，miR-26可能通过调节ddx3x的表达在medaka繁殖发育中发挥重要作用。双荧光素酶报告基因测定法和miR-26 agomir和antagomir的注射证明Olddx3x的表达受miR-26调控，但不受miR-202-5p调控。总体结果表明，miR-26可能通过调节ddx3x的表达在medaka繁殖发育中发挥重要作用。